This invention relates to 1,4,7,10-tetraazacyclodedecane butyltriols, their complexes and complex salts, agents containing these compounds, their use as diagnostic agents and therapeutic agents as well as processes for the production of these compounds and agents.
In European patent application 87730085.5 with the publication number 0 255 471 macrocyclic compounds of general formula I 
are claimed, in which
Y is a nitrogen atom or phosphorus atom,
A1 and A2 are the same or different and each is a straight-chain or branched alkylene group with 2 to 6 carbon atoms,
U1, U2, U3, U4 are the same or different and each is a direct bond or a straight-chain or branched alkylene group with 1 to 6 carbon atoms,
D1, D2, D3, D4 are the same or different and each is an oxygen atom or sulfur atom, an alkylene group with 1 to 6 carbon atoms or a group N-R7 with R7 meaning a hydrogen atom, a straight-chain or branched alkylene chain with 1 to 4 carbon atoms, which on the end carries a COOR1 group, and R1 stands for a hydrogen atom or a metal ion equivalent.
D5 has the meaning indicated for D1, D2, D3 and D4 as well as the group 
xe2x80x83with R5 meaning a hydrogen atom or a straight-chain or branched saturated or unsaturated C1-C20 alkylene group, optionally substituted by hydroxy, mercapto, imino and/or amino group(s), optionally containing imino, phenylenoxy, phenylenimino, amide, ester group(s), oxygen atom(s), sulfur atom(s) and/or nitrogen atom(s), which on the end exhibits either a functional group or a macromolecule B bound by it,
s and t represent whole numbers from 0 to 5,
R2 represents hydrogen, a straight or branched, saturated or unsaturated alkyl, acyl or acylalkyl group with 1 to 16 carbon atoms optionally substituted by one or more hydroxy or lower alkoxy groups,
xe2x80x94CH2xe2x80x94Xxe2x80x94V with X meaning carbonyl, a straight-chain or branched-chain alkylene group with 0 to 10 carbon atoms, which optionally is substituted by one or more hydroxy or lower alkoxy groups or a straight-chain or branched-chain alkylene group interrupted by oxygen atoms with 2 to 23 hydrocarbon atoms, 
xe2x80x83and R3 and R4, independently
xe2x80x83independently of one another, represent hydrogen, a straight or branched alkyl group with 1 to 16 carbon atoms optionally substituted by one or more hydroxy or lower alkoxy groups or R3 and R4 together with the nitrogen atom represent a saturated five or six ring optionally containing another heteroatom and R6 represents hydrogen or a saturated, unsaturated straight-chain or branched-chain or cyclic hydrocarbon radical with up to 16 carbon atoms or an aryl or aralkyl group,
xe2x80x83or
R2 or R3 represent a second macrocycle of formula Ixe2x80x2xe2x80x94bound by an alkylene chain (K) containing 2 to 20 carbon atoms, which optionally carries carbonyl groups on the ends and optionally is interrupted by one or more oxygen atoms or R1 carboxymethylimino groups or is substituted by one or more hydroxy, lower alkoxy or carboxy lower alkyl groupsxe2x80x94
which can be of a different structure than the parent substance of the first, or
R2 means B or CH2xe2x80x94COB,
provided that, if R2 stands for B or CH2xe2x80x94COB, R5 means a hydrogen atom, that at least two COOR1 groups are present in the molecule and that two heteroatoms of the macrocycle are each connected by an alkylene group with at least two carbon atoms, and functional groups existing in the molecule optionally are conjugated with macromolecules and optionally free carboxyl groups are made into salts with organic or inorganic bases or amino acids and basic groups with inorganic or organic acids.
The substances and the solutions prepared from them meet the demands to be made of pharmaceutically usable chelates. They have a strong and adaptable effectiveness, by the selection of suitable metal atoms, on the respective principles of the diagnostic or therapeutic method (x ray, NMR, ultrasound, nuclear medicine).
Of the many compounds in EP-A-0 255 471, the 1,4,7,10-tetraazacyclodedecane butyltriols of general formula IA 
in which
R1 means hydrogen or a metal ion equivalent independent of one another and
R2 means a butyltriol radical
as well as their salts with organic or inorganic bases or amino acids, exhibit such outstanding properties that even in comparison with the structurally very closely related compound disclosed in EP-A 0 255 471 (Example 6), the use of these selected compounds guarantees a substantial advantage.
Compounds of general formula IA with R1 meaning hydrogen are designated as complexing agents and with at least two of substituents R1 meaning a metal ion equivalent are designated as metal complexes.
The element, which forms the central ion of the physiologically compatible complex salt, can, of course, also be radioactive for desired purpose of use of the diagnostic medium according to the invention.
If the medium according to the invention is intended for use in NMR diagnosis, the central ion of the complex salt has to be paramagnetic. This involves especially the bivalent and trivalent ions of the elements of the atomics numbers 21-29, 42, 44 and 58-70. Suitable ions are, for example, the chromium(III), manganese(II), iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III) and ytterbium(III) ion. Because of their very strong magnetic moment there are especially preferred the gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III) or iron(III) ion.
For use of the media according to the invention in nuclear medicine the central ion has to be radioactive. For example, radioisotopes of the elements copper, cobalt, gallium. germanium, yttrium, strontium, technetium, indium, ytterbium, gadolinium, samarium and iridium are suitable.
If the medium according to the invention is intended for use in x-ray diagnosis, the central ion has to be derived from an element of higher atomic number to achieve a sufficient absorption of the x rays. It was found that for this purpose diagnostic media, which contain a physiologically compatible complex salt with central ions of elements of the atomic numbers between 21-29, 42, 44, 57-83, are suitable; they are, for example, the lanthanum(III) ion and the above-named ions of the lanthanide series.
Preferred radicals R2 are 2,3,4-trihydroxybutyl- and the 1-hydroxymethyl-2,3-dihydroxypropyl radical.
If not all acidic hydrogen atoms are substituted by the central ion, one, several or all remaining hydrogen atom(s) can be replaced by cations of inorganic and/or organic bases or amino acids. Suitable inorganic cations are, for example, the lithium ion, the potassium ion, the calcium ion, the magnesium ion and especially the sodium ion. Suitable cations of organic bases are, among others, those of primary, secondary or tertiary amines, such as, for example, ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethylglucamine and especially N-methylglucamine. Suitable cations of amino acids are, for example, those of lysine, arginine and ornithine.
As proof for the above-named surprising and outstanding properties of the compounds according to the invention there are to be indicated the results of animal experimental studies to determine the acute intravenous (LD50) as well as neural compatibility (ED50) of two butyltriol compounds according to the invention, i.e., of compounds of general formula IA in comparison with the compound described in EP-A 0 255 471, which structurally is closest to the two butyltriol macrocycles:
1. Acute Toxicity Determination (LD50)
In an individual cage (Rhema/Hofheim company) the contrast medium was administered to mice (weight: 18-22 g) approximately at body temperature in a caudal vein at a rate of 2 ml/min. Also preferred are those of 10-[2-hydroxy-2,2-bis(hydroxymethyl)ethyl)]-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane, and 10-[1,1,1-tris(hydroxymethyl)methyl]-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane.
The contrast media were administered to 3 mice each in 3 doses at variable volumes and constant concentration. The allocation of the animals to the individual doses and the injection sequence of the doses took place by chance.
The observation period of the animals extended over 7 days per injection. The death of the animals was used as the criterion of the effectiveness.
2. Intracisternal Administration (ED50)
The test substance was administered under light ether anesthesia to an equal number of female and male rats (130-170 g, Wistar-Han-Schering, SPF) once intracisternally by suboccipital puncture in constant volumes (0.08 ml/animal) in different concentrations (3 doses per 10 animals per substance). A volume sampling check (Ringer""s solution) followed the study. Posture anomalies, spasms and death of the animals were evaluated as action criteria.
The statistical evaluation of all study results took place with the probit analysis.
3. Results
Contrast media for medical diagnosis should be eminently compatible and behave in as biologically inert manner as possible. Monomer contrast media should come as close as possible to the intravenous compatibility of, e.g., mannitol (i.e., 30-40 mmol/kg). Comparable compounds with lower values therefore are biologically not inert and show undesirable interaction with the organism. The propanediol derivative from EP-A 0 255 471 shows with an acute i.v. LD50 of 25 mmol/kg clearly lower values than the two compounds according to the invention. This relevant difference indicates a certain chemotoxicity of the comparison substance. The undesirable interaction was shown particularly in the comparison of the test substances in the neural compatibility. The two compounds according to the invention showed a clearly better compatibility. With the propanediol compound already at doses of 14 micromol/kg changes in the behavior and marked spasms occurred. Some animals died at this dose.
With the macrocyclic gadolinium complexes of this invention a substantially better neural compatibility could be observed. A detrimental effect on behavior occurred only with doses approximately twice as high as with the comparison substance.
Conclusion
Since the two butyltriol compounds according to the invention also show a lower osmolality (0.59 or 0.57 in comparison with 0.62 osmol/kg; aqueous solutions of the Gd Complexes in a concentration of 0.5 mol/l were measured) than the above-described propanediol compound, in summary it can be noted that the undesirable interactions of this compound with the biological organism surprisingly do not occur with the structurally closely related compounds according to the invention. The two butyltriol compounds show marked advantages and therefore are substantially more suitable as biologically inert contrast media.
The production of the compounds of general formula IA according to the invention takes place in that compounds of general formula II 
in which
X stands for a nitrogen protecting group or CH2COOY group with Y meaning hydrogen, an ammonium cation, an alkali metal or a protecting group,
are reacted with a substrate introducing the radical R2 in protected form, the optionally contained nitrogen protecting groups X are removed and the xe2x80x94NHxe2x80x94 groups thus released are alkylated with an acetic acid derivative of general formula III
HalCH2COOYxe2x80x83xe2x80x83(III)
in which Hal stands for chlorine, bromine or iodine,
the (hydroxy and optionally acid) protecting groups are removed and the resulting compounds of general formula IA with R1 meaning hydrogen are converted with metal oxide or metal salt into the metal complexes of general formula IA with R1 meaning a metal ion equivalent and thenxe2x80x94if desiredxe2x80x94still existing acidic hydrogen atoms are substituted by cations of inorganic and/or organic bases, amino acids or amino acid amides.
Suitable as acid protecting groups Y are lower alkyl, aryl and aralkyl groups, for example, the methyl, ethyl, propyl, n-butyl, t-butyl, phenyl, benzyl, diphenylmethyl, triphenylmethyl, bis(p-nitrophenyl)-methyl group as well as trialkylsilyl groups.
The acids HalCH2COOH can also be used in the form of their salts, preferably as Na or K salt.
The cleavage of the acid protecting groups takes place according to processes known to one skilled in the art, for example, by hydrolysis, hydrogenolysis, alkaline saponification of the esters with alkali in aqueous alcoholic solution at temperatures of 0 to 50xc2x0 C., acid saponification with mineral acids or in case, e.g., of tert-butyl esters with the help of trifluoroacetic acid.
The three nitrogen atoms of feedstock II carry, before the reaction introducing the radical R2, the group CH2COOY or nitrogen protecting groups, for example, the tosylate or trifluoroacetate group, which are cleaved according to methods known in the literature before the alkylation reaction that is to follow [the tosylates, e.g., with mineral acids, alkali metals in liquid ammonia, hydrobromic acid and phenol, RedAl(R), lithiumaluminum hydride, sodium amalgam, cf. e.g, Liebigs Ann. Chem. (1977), 1344, Tetrahedron Letters (1976), 3477; the trifluoracetates, e.g. with mineral acids or ammonia in methanol, cf.e.g., Tetrahedron Letters (1967), 289].
The N-alkylation with a haloacetic acid derivative of general formula III takes place in polar aprotic solvents such as, for example, dimethylformamide, dimethyl sulfoxide, acetonitrile, aqueous tetrahydrofuran or hexamethylphosphoric acid triamide in the presence of an acid trap such as, for example, tertiary amine (for example, triethylamine, trimethylamine, N,N-dimethylaminopyridine, 1,5-diazabicyclo[4.3.0]-nonene-5(DBN), 1,5-diazobicyclo[5.4.0]-undecene-5-(DBU), alkali, alkaline-earth carbonate, bicarbonate or hydroxide (for example, lithium, sodium, magnesium, calcium, barium, potassium carbonate, hydroxide and bicarbonate) at temperatures between xe2x88x9210xc2x0 C. and 120xc2x0 C., preferably between 0xc2x0 C. and 50xc2x0 C.
Suitable as hydroxy protecting groups are all those that can easily be introduced and later again be easily cleaved with re-formation of the finally desired free hydroxy group. Preferred protecting groups are ether groups such as, for example, the benzyl, 4-methoxybenzyl, 4-nitrobenzyl, trityl, di- and tri-phenylmethyl, trimethylsilyl, dimethyl-t-butylsilyl, diphenyl-t-butylsilyl group. But preferably the hydroxy groups are protected in the form of ketals, for example, with acetone, acetaldehyde, cyclohexanone or benzaldehyde.
The cleavage of the hydroxy protecting groups takes place in a way known in the art, for example, in case of a benzyl ether by reductive cleavage with lithium/ammonia or by hydrogenolytic cleavage in the presence, for example, of palladium-carbon and in case of a ether or ketal cleavage by acid treatment with the help, for example, of cation exchangers, trifluoroacetic acid or mineral acids [see, e.g., T. W. Greene xe2x80x9cProtective Groups in Organic Synthesis,xe2x80x9d John Wiley and Sons (1981)].
The introduction of the radical R2 takes place by alkylation of a substrate synthesized from a nucleofuge group for example Cl, Br, I, CH3C6H4SO3, CF3SO3, CH3SO3 and of the protected radical R2 or of a substrate from which the desired radical R2 is intramolecularly generated during the reaction. As an example for the last mentioned case there can be mentioned, the hydroxy epoxides 2,3-epoxy-1,4-dihydroxybutane and 1,2-epoxy-3,4-dihydroxybutane protected as, e.g., acetonides.
The reaction of feedstock II with X meaning a CH2COOY group is performed, e.g., in water, DMF, dioxane, alcohols, acetonitrile, tetrahydrofuran or their mixtures at temperatures of 0 to 100xc2x0 C., preferably room temperature to 60xc2x0 C., at a basic pH, preferably 9 to 13, within 6 hours to 2 days, preferably 12 to 36 hours.
If a macrocycle, protected on the residual nitrogen atoms, is used for the introduction of the R2 radical in the reaction, the reaction takes place preferably in an autoclave in solvents such as, for example, DMF, DMA, toluene, methylene chloride or their mixtures at temperatures of 20 to 170xc2x0 C., preferably 100 to 150xc2x0 C., with addition of a base, such as, e.g., amines, alkali, alkaline-earth hydroxides and carbonates, preferably potassium and sodium carbonate and hydroxide, within 6 hours to 2 days, preferably 12 to 36 hours. If a substrate is used, which contains no nucleofuge group (i.e., for example, the above-mentioned epoxides), the use of a base can be dispensed with.
The compounds of general formula IA with R1 meaning a hydrogen atom represent complexing agents. They can be isolated and purified or without isolation can be converted into metal complexes of general formula Ia with at least two of substituents R1 meaning a metal ion equivalent.
The production of the metal complexes according to the invention takes place in the way that was disclosed in German laid-open specification 34 01 052, by the metal oxide or a metal salt (for example, the nitrate, acetate, carbonate, chloride or sulfate of the element of the atomic numbers 21-29, 42, 44, 57-83) being dissolved or suspended in water and/or a lower alcohol (such as methanol, ethanol or isopropanol) and being reacted with the solution or suspension of the equivalent amount of the complexing ligand and then, if desired, by existing acidic hydrogen atoms being substituted by cations of inorganic and/or organic bases or amino acids.
The introduction of the desired metal ions take place both before and after cleavage of the hydroxy protecting groups.
The neutralization of possibly still existing free carboxy groups takes place with the help of inorganic bases (for example, hydroxides, carbonates or bicarbonates), for example, of sodium, potassium, lithium, magnesium or calcium and/or organic bases as, among others, primary, secondary and tertiary amines, such as, for example, ethanolamine, morpholine, glucamine, N-methyl and N,N-dimethylglucamine, as well as basic amino acids, such as, for example, lysine, arginine and ornithine or of amides of originally neutral or acidic amino acids.
For the production of neutral complex compounds so much of the desired bases is added to the acid complex salts in aqueous solution or suspension that the neutral point is reached. The resulting solution can then be evaporated to dryness in a vacuum. Often it is advantageous to precipitate the formed neutral salts by addition of water-miscible solvents, such as, for example, lower alcohols (methanol, ethanol, isopropanol and others), lower ketones (acetone and others), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoxyethane and others) and thus obtain crystallizates that are easy to isolate and purify. It has proved especially advantageous to add the desired base already during the complex formation of the reaction mixture and thus to save a process step.
Another possibility to achieve neutral complex compounds consists in converting the remaining acid groups in the complex entirely or partially, for example, to esters or amides. This can occur by subsequent reaction on the finished complex (e.g., by exhaustive reaction of the free carboxy groups with dimethyl sulfate.
The production of the pharmaceutical agents according to the invention also takes place in a way known in the art, by complex compounds according to the inventionxe2x80x94optionally by addition of additives usual in galenicalsxe2x80x94being suspended or dissolved in aqueous medium and then the suspension or solution optionally being sterilized. Suitable additives are, for example, physiologically safe buffers (such as, for example, tromethamine), additives of complexing agents (such as, for example, diethylenetriaminepentaacetic acid) orxe2x80x94if desiredxe2x80x94electrolytes, such as, for example, sodium, calcium, magnesium, zinc chlorides, phosphates and citrates orxe2x80x94if necessaryxe2x80x94antioxidizing agents, such as, for example, ascorbic acid.
If suspensions or solutions of agents according to the invention in water or physiological salt solution are desired for enteral administration or other purposes, they are mixed with one or more auxiliary agents usual in galenicals (for example, methylcellulose, lactose, mannitol) and/or surfactants (for example, lecithins, Tween(R), myrj(R)) and/or aromatic substance(s) for taste correction (for example, essential oils).
In principle it is also possible to produce pharmaceutical agents according to the invention even without isolation of complex salts. In any case, special care must be used to perform the chelate formation so that the salts and salt solutions according to the invention are produced practically free of uncomplexed toxically acting metal ions.
This can be guaranteed, for example with the help of color indicators such as xylenol orange by control filtrations during the production process. The invention therefore relates also to processes for the production of complex compounds and their salts. A purification of the isolated complex salt remains as final safety measure.
The pharmaceutical agents according to the invention preferably contain 0.1 micromol-3 mol/l of the complex salt and generally are dosed in amounts of 0.1 micromol-5 mmol/kg. They are intended for enteral and parenteral administration. The complex compounds according to the invention can be used:
1. for NMR and x-ray diagnosis in the form of their complexes with the ions of elements without atomic numbers 21-29, 42, 44 and 57-83;
2. for radiodiagnosis and radiotherapy in the form of their complexes with the radioisotopes of the elements with atomic numbers 27, 29, 31, 32, 37-39, 43, 49, 62, 64, 70, 75 and 77.
The agents according to the invention meet the varied requirements for the suitability as contract media for nuclear spin tomography. Thus they are outstandingly suitable for improving, in its expressive power, the image obtained with the nuclear spin tomograph, by enhancing the signal intensity after enteral or parenteral administration. Further, they show the great effectiveness, which is necessary, to load the body with the smallest possible amounts of foreign substances, and the good compatibility, which is necessary to maintain the noninvasive character of the examinations.
The good water solubility and low osmolality of the agents according to the invention make it possible to produce highly concentrated solutions, to maintain the volume load of the circulation in justifiable limits and to balance the dilution by the body fluid, i.e., NMR diagnostic media have to be 100 to 1000 times better water soluble than for NMR spectroscopy. Further, the agents according to the invention exhibit not only a great stability in vitro but also a surprisingly great stability in vivo so that a release or an exchange of the ions, not covalently bound in the complexesxe2x80x94toxic in themselvesxe2x80x94within the time, in which the new contrast media again are completely excreted now takes place extremely slowly.
In general, the agents according to the invention for use as NMR diagnostic media was dosed in amounts of 0.0001-5 mmol/kg, preferably 0.005-05 mmol/kg. Details of the use are discussed, for example, in H. J. Weinmann et al., Am. J. of Roentgenology 142, 619 (1984).
Further, the complex compounds according to the invention can advantageously be used as susceptibility reagents and shift reagents for the in vivo NMR spectroscopy.
Because of their favorable radioactive properties and of the good stability of the complex compounds contained in them, the agents according to the invention are also suitable as radiodiagnostic media. Details of their use and dosage are described, e.g., in xe2x80x9cRadiotracers for Medical Applications,xe2x80x9d CRC Press, Boca Raton, Fla.
Another imaging method with radioisotopes is positron emission tomography, which uses positron-emitting isotopes such as, for example, 43Sc, 44Sc, 52Fe, 55Co and 68Ga (Heiss, W. D.; Phelps, M. E.; Positron Emission Tomography of Brain, Springer Verlag Berlin, Heidelberg, N.Y. 1983).
The compounds according to the invention can also be used in radioimmuno or radiation therapy. It differs from the corresponding diagnosis only by the amount and type of isotope used. In this case, the aim is to destroy tumor cells by energy-rich shortwave radiation with a smallest possible range. Suitable beta-emitting ions are, for example, 46Sc, 47Sc, 48Sc, 72Ga, 73Ga and 90Y. Suitable alpha-emitting ions exhibiting a short half-life are, for example, 211Bi, 212Bi, 213Bi and 214Bi, and 212Bi is preferred. A suitable photon- and electron-emitting ion is 158Gd, which can be obtained from 157Gd by neutron capture.
If the agent according to the invention is intended for the use in the variant of radiation therapy proposed by R. L. Mills et al. [Nature Vol. 336 (1988), p. 787], the central ion has to be derived from a Moessbauer isotope such as, for example, 57Fe or 151Eu.
In the in vivo application of the therapeutic agents according to the invention, they can be administered together with a suitable carrier such as, for example, serum or physiological common salt solution and together with another protein such as, for example, human serum albumin. In this case, the dosage depends on type of cellular impairment, the metal ion used and the type of method, e.g., brachytherapy.
The therapeutic agents according to the invention are administered parenterally.
Details of the use of radiotherapeutic agents are discussed, e.g., in R. W. Kozak et al. TIBTEC, October 1986, 262.
The agents according to the invention, because of their outstanding water solubility and because of the low osmotic pressure of their concentrated aqueous solutions, are excellent x-ray contrast media, and it is to be especially emphasized that with them in biochemical-pharmacological studies no indications can be perceived of the known anaphylactic-type reactions from the iodine-containing contrast media. Because of the favorable absorption properties in the ranges of higher tube voltages they are especially valuable for digital subtraction techniques.
In general, the agents according to the invention for use as x-ray contrast media are dosed analogously to, e.g., meglumine diatrizoate in amounts of 0.1-5 mmol/kg, preferably 0.25-1 mmol/kg.
Details of the use of x-ray contrast media are discussed, for example, in Barke, Roentgenkontrastmittel [X-ray Contrast Media], G. Thieme, Leipzig (1970) and P. Thurn, E. Buechelerxe2x80x94Einfuehrung in die Roentgendiagnostik [Introduction to X-ray Diagnosis], G. Thieme, Stuttgart, N.Y. (1977).
Altogether it has been possible to synthesize new complexing agents, metal complexes and metal complex salts, which open up new possibilities in diagnostic and therapeutic medicine. Especially the development of novel imaging processes in medical diagnosis makes this development appear desirable.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.
The entire disclosure of all applications, patents and publications, cited above and below, and of corresponding German application P 40 09 119.8 are hereby incorporated by reference.